Variants of chymosin with improved milk-clotting properties

ABSTRACT

Variants of chymosin with improved milk clotting properties.

FIELD OF THE INVENTION

The present invention relates to variants of chymosin with improvedmilk-clotting properties.

BACKGROUND ART

Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clottingenzymes of the mammalian stomach, are aspartic proteases belonging to abroad class of peptidases.

When produced in the gastric mucosal cells, chymosin and pepsin occur asenzymatically inactive pre-prochymosin and pre-pepsinogen, respectively.When chymosin is excreted, an N-terminal peptide fragment, thepre-fragment (signal peptide) is cleaved off to give prochymosinincluding a pro-fragment. Prochymosin is a substantially inactive formof the enzyme which, however, becomes activated under acidic conditionsto the active chymosin by autocatalytic removal of the pro-fragment.This activation occurs in vivo in the gastric lumen under appropriate pHconditions or in vitro under acidic conditions.

The structural and functional characteristics of bovine, i.e. Bostaurus, preprochymosin, prochymosin and chymosin have been studiedextensively. The pre-part of the bovine pre-prochymosin moleculecomprises 16 aa residues and the pro-part of the correspondingprochymosin has a length of 42 aa residues. The active bovine chymosincomprises 323 aa.

Chymosin is produced naturally in mammalian species such as bovines,camels, caprines, buffaloes, sheep, pigs, humans, monkeys and rats.

Bovine and camel chymosin has for a number of years been commerciallyavailable to the dairy industry.

Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosinand pepsin, is one of the most important processes in the manufacture ofcheeses. Enzymatic milk coagulation is a two-phase process: a firstphase where a proteolytic enzyme, chymosin or pepsin, attacks κ-casein ,resulting in a metastable state of the casein micelle structure and asecond phase, where the milk subsequently coagulates and forms acoagulum (reference 1).

WO02/36752A2 (Chr. Hansen) describes recombinant production of camelchymosin.

WO2013/174840A1 (Chr. Hansen) describes mutants/variants of bovine andcamel chymosin.

WO2013/164479A2 (DSM) describes mutants of bovine chymosin.

The references listed immediately below may in the present context beseen as references describing mutants of chymosin:

-   -   Suzuki et al: Site directed mutagenesis reveals functional        contribution of Thr218, Lys220 and Asp304 in chymosin, Protein        Engineering, vol. 4, January 1990, pages 69-71;    -   Suzuki et al: Alteration of catalytic properties of chymosin by        site-directed mutagenesis, Protein Engineering, vol. 2, May        1989, pages 563-569;    -   van den Brink et al: Increased production of chymosin by        glycosylation, Journal of biotechnology, vol. 125, September        2006, pages 304-310;    -   Pitts et al: Expression and characterisation of chymosin pH        optima mutants produced in Tricoderma reesei, Journal of        biotechnology, vol. 28, March 1993, pages 69-83;    -   M. G. Williams et al: Mutagenesis, biochemical characterization        and X-ray structural analysis of point mutants of bovine        chymosin, Protein engineering design and selection, vol. 10,        September 1997, pages 991-997;    -   Strop et al: Engineering enzyme subsite specificity:        preparation, kinetic characterization, and x-ray analysis at 2.0        ANG resolution of Va1111phe site mutated calf chymosin,        Biochemistry, vol. 29, October 1990, pages 9863-9871;    -   Chitpinityol et al: Site-specific mutations of calf chymosin B        which influence milk-clotting activity, Food Chemistry, vol. 62,        June 1998, pages 133-139;    -   Zhang et al: Functional implications of disulfide bond,        Cys45-Cys50, in recombinant prochymosin, Biochimica et        biophysica acta, vol. 1343, December 1997, pages 278-286.

None of the prior art references mentioned above describe directly andunambiguously any of the chymosin variants with improved specificclotting activity or increased C/P ratios compared to the parent fromwhich the variant is derived, as described below.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide variantsof chymosin which when compared to the parent polypeptide, have eitherincreased specific clotting activity or increased C/P ratio or both.

Based on intelligent design and comparative analyses of differentvariants, the present inventors identified a number of amino acidpositions that are herein important in the sense that by making avariant in one or more of these positions in a parent peptide one mayget an improved chymosin variant with either increased specific clottingactivity or increased C/P ratios or both.

The amino acid numbering as used herein to specify the variant is basedon the mature peptide. As known in the art—different natural wildtypechymosin polypeptide sequences obtained from different mammalian species(such as e.g. bovines, camels, sheep, pigs, or rats) are having arelatively high sequence similarity/identity. In FIG. 1 this isexemplified by an alignment of herein relevant different chymosinsequences.

In view of this relatively close sequence relationship—it is believedthat the 3D structures of different natural wildtype chymosins are alsorelatively similar.

In the present context—a naturally obtained wildtype chymosin (such asbovine chymosin or camel chymosin) may herein be an example of a parentpolypeptide—i.e. a parent polypeptide to which an alteration is made toproduce a variant chymosin polypeptide of the present invention.

Without being limited to theory—it is believed that the herein discussedchymosin related amino acid positions are of general importance in anyherein relevant chymosin enzyme of interest (e.g. chymosins of e.g.bovines, camels, sheep, pigs, rats etc.)—in the sense that by making avariant in one or more of these positions one may get an improvedchymosin variant in general (e.g. an improved bovine, camel, sheep, pigor rat chymosin variant).

As discussed herein—as a reference sequence for determining the aminoacid position of a parent chymosin polypeptide of interest (e.g. camel,sheep, bovine etc.) is herein used the public known Camelius dromedariusmature chymosin sequence of SEQ ID NO:2 herein. It may hereinalternatively be termed camel chymosin. The sequence is also shown inFIG. 1 herein.

In the present context it is believed that a parent chymosin polypeptide(e.g. rom sheep or rat) that has at least 80% sequence identity with themature polypeptide of SEQ ID NO:2 (camel chymosin) may herein be seen assufficient structural related to e.g. bovine or camel chymosin in orderto be improved by making a variant in any of the amino acid positions asdescribed herein.

Embodiments of the present invention are described below.

DEFINITIONS

All definitions of herein relevant terms are in accordance of what wouldbe understood by the skilled person in relation to the herein relevanttechnical context.

The term “chymosin” relates to an enzyme of the EC 3.4.23.4 class.Chymosin has a high specificity and predominantly clots milk by cleavageof a single 104-Ser-Phe-|-Met-Ala-107 bond in κ-chain of casein. As aside-activity, chymosin also cleaves α-casein primarily between Phe23and Phe24 and β-casein primarily between Leu192 and Tyr193 (references2, 3). The resulting peptides αS1(1-23) and β (193-209) will be furtherdegraded by proteases from microbial cultures added to the ripeningcheese (reference 4). An alternative name of chymosin used in the art isrennin.

The term “chymosin activity” relates to chymosin activity of a chymosinenzyme as understood by the skilled person in the present context.

The skilled person knows how to determine herein relevant chymosinactivity.

As known in the art—the herein relevant so-called C/P ratio isdetermined by dividing the specific clotting activity (C) with theproteolytic activity (P). As known in the art—a higher C/P ratio impliesgenerally that the loss of protein during e.g. cheese manufacturing dueto non-specific protein degradation is reduced which may lead to cheeseyield improvements.

The term “isolated variant” means a variant that is modified by the actof man. In one aspect, the variant is at least 1% pure, e.g., at least5% pure, at least 10% pure, at least 20% pure, at least 40% pure, atleast 60% pure, at least 80% pure, and at least 90% pure, as determinedby SDS PAGE.

The term “mature polypeptide” means a peptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In the present context may a herein relevantmature chymosin polypeptide be seen as the active chymosin polypeptidesequence—i.e. without the pre-part and/or pro-part sequences. Hereinrelevant examples of a mature polypeptide are e.g. the maturepolypeptide of SEQ ID NO:1 (bovine chymosin), which is from amino acidposition 59 to amino acid position 381 of SEQ ID NO:1 or the maturepolypeptide of SEQ ID NO:2 (camel chymosin), which is from amino acidposition 59 to amino acid position 381 of SEQ ID NO:2.

The term “parent” or “parent polypeptide having chymosin activity” meansa polypeptide to which an alteration is made to produce the enzymevariants of the present invention. The parent may be a naturallyoccurring (wild-type) polypeptide or a variant thereof.

The term “Sequence Identity” relates to the relatedness between twoamino acid sequences or between two nucleotide sequences.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

The term “variant” means a peptide having chymosin activity comprisingan alteration, i.e., a substitution, insertion, and/or deletion, at oneor more (several) positions. A substitution means a replacement of anamino acid occupying a position with a different amino acid; a deletionmeans removal of an amino acid occupying a position; and an insertionmeans adding 1-3 amino acids adjacent to an amino acid occupying aposition.

The amino acid may be natural or unnatural amino acids—for instance,substitution with e.g. a particularly D-isomers (or D-forms) of e.g.D-alanine could theoretically be possible.

The term “wild-type” peptide refers to a nucleotide sequence or peptidesequence as it occurs in nature, i.e. nucleotide sequence or peptidesequence which hasn't been subject to targeted mutations by the act ofman.

DRAWINGS

FIG. 1: An alignment of herein relevant different chymosin sequences.

As understood by the skilled person in the present context—hereinrelevant sequence identity percentages of mature polypeptide sequencesof e.g. sheep, C. bactrianus, camel, pig or rat chymosin with the maturepolypeptide of SEQ ID NO:3 (bovine chymosin—i.e. amino acid positions 59to 381 of SEQ ID NO:3) are relatively similar to above mentionedsequence identity percentages.

FIG. 2:

3D structure of camel chymosin (detail, PDB: 4AA9) with a model of boundκ-casein shown in green. κ-casein is placed in the chymosin substratebinding cleft with the scissile bond between residues 105 and 106.Mutations R242E, Y243E, N249D, G251D, N252D, R254E, S273D, Q280E, F282Eare highlighted in blue.

FIG. 3:

3D structure of bovine chymosin (PDB: 4AA8) with a model of boundκ-casein shown in green. κ-casein is placed in the chymosin substratebinding cleft with the scissile bond between residues 105 and 106.Positions H292 and Q294 are highlighted in yellow.

FIG. 4:

3D structure of camel chymosin (detail, PDB: 4AA9). Residues Y11, L12,and D13 of the protein N-terminus as well as the potential Y11interaction partner D290 are highlighted in purple.

DETAILED DESCRIPTION OF THE INVENTION

Determining the Amino Acid Position of a Chymosin of Interest

As discussed above—as a reference sequence for determining the aminoacid position of a herein relevant chymosin polypeptide of interest(e.g. camel, sheep, bovine etc.) is herein used the public known camelchymosin sequence disclosed as SEQ ID NO:2 herein.

The amino acid sequence of another chymosin polypeptide is aligned withthe polypeptide disclosed in SEQ ID NO:2, and based on the alignment,the amino acid position number corresponding to any amino acid residuein the polypeptide disclosed in SEQ ID NO:2 is determined using theClustalW algorithm as described in working Example 1 herein.

Based on above well-known computer programs—it is routine work for theskilled person to determine the amino acid position of a herein relevantchymosin polypeptide of interest (e.g. camel, sheep, bovine etc.).

In FIG. 1 herein is shown an example of an alignment.

Just as an example—in FIG. 1 can e.g. be seen that herein used bovinereference SEQ ID NO:3 has a “G” in position 50 and “Camelus_dromedarius”(SEQ ID NO:2 herein) has an “A” in this position 50.

Nomenclature of Variants

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviations are employed.

The specific variants discussed in this “nomenclature” section below maynot be herein relevant variants of the present invention—i.e. this“nomenclature” section is just to describe the herein relevant usednomenclature as such.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, a theoretical substitution of threonine with alanineat position 226 is designated as “Thr226Ala” or “T226A”. Multiplemutations are separated by addition marks (“+”), e.g.,“Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions atpositions 205 and 411 of glycine (G) with arginine (R) and serine (S)with phenylalanine (F), respectively. A substitution e.g. designated“226A” refers to a substitution of a parent amino acid (e.g. T, Q, S oranother parent amino acid) with alanine at position 226.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Glyl95*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Glyl95*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Orignal amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Glyl95GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Glyl95GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Glyl95Glu” or“R170Y+G195E” representing a substitution of tyrosine and glutamic acidfor arginine and glycine at positions 170 and 195, respectively.

Different substitutions. Where different substitutions can be introducedat a position, the different substitutions are separated by a comma,e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of argininewith tyrosine or glutamic acid at position 170. Thus,“Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

Preferred Parent Polypeptide having Chymosin Activity

Preferably, the parent polypeptide has at least 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% sequence identity with the mature polypeptide of SEQ IDNO:3 (bovine chymosin) and/or SEQ ID NO:2 (camel chymosin).

Just as an example—a herein suitable relevant parent polypeptide coulde.g. be bovine chymosin A—as known in the art bovine chymosin A may onlyhave one amino acid difference as compared to bovine chymosin B of SEQID NO:3 herein.

In a preferred embodiment—the parent polypeptide has at least 90%sequence identity with the mature polypeptide of SEQ ID NO:3 (bovinechymosin), more preferably the parent polypeptide has at least 95%sequence identity with the mature polypeptide of SEQ ID NO:3 (bovinechymosin) and even more preferably the parent polypeptide has at least97% sequence identity with the mature poly-peptide of SEQ ID NO:3(bovine chymosin). It may be preferred that the parent polypeptide isthe mature polypeptide of SEQ ID NO:3 (bovine chymosin).

As understood by the skilled person in the present context—a hereinrelevant parent polypeptide having chymosin activity may already e.g. bea variant of e.g. a corresponding wildtype chymosin.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to mature wildtype bovine chymosinpolypeptide of SEQ ID NO:3 may still be a parent polypeptide that has atleast 95% sequence identity with the mature polypeptide of SEQ ID NO:3(Bovine chymosin).

Said in other words and in general—a herein relevant isolated chymosinpoly-peptide variant may comprise alterations (e.g. substitutions) inother positions than the positions claimed herein.

As understood by the skilled person in the present context—a parentpolypeptide may be a polypeptide that has at least 80% sequence identitywith the mature polypeptide of SEQ ID NO:2 (Camel). In a preferredembodiment—the parent polypeptide has at least 92% sequence identitywith the mature polypeptide of SEQ ID NO:2 and/or SEQ ID NO:3, morepreferably the parent polypeptide has at least 95% sequence identitywith the mature polypeptide of SEQ ID NO:2 and/or SEQ ID NO:3 and evenmore preferably the parent polypeptide has at least 97% sequenceidentity with the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:3. Itmay be preferred that the parent polypeptide is the mature polypeptideof SEQ ID NO:2 (camel chymosin).

As understood by the skilled person in the present context—an isolatedchymosin variant may comprise alterations (e.g. substitutions) in otheramino acid positions than given above.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to wildtype camel chymosin polypeptide of SEQID NO:2 will still be a parent polypeptide that has at least 95%sequence identity with the mature polypeptide of SEQ ID NO:2.

It may be preferred that the isolated bovine chymosin variant comprisesless than 30 amino acid alterations (e.g. substitutions) as compared tothe mature polypeptide of SEQ ID NO:2 (camel chymosin) or it may bepreferred that the isolated camel chymosin variant comprises less than20 amino acid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO:2 or it may be preferred that the isolatedbovine chymosin variant comprises less than 10 amino acid alterations(e.g. substitutions) as compared to the mature polypeptide of SEQ IDNO:2 or it may be preferred that the isolated camel chymosin variantcomprises less than 5 amino acid alterations (e.g. substitutions) ascompared to the mature polypeptide of SEQ ID NO:2 (camel chymosin).

Method for Making Isolated Chymosin Polypeptide Variants

As discussed above—as known in the art, the skilled person may, based onhis common general knowledge, routinely produce and purify chymosin andchymosin variants.

Said in other words, once the skilled person is in possession of aherein relevant parent polypeptide having chymosin activity of interest(e.g. from bovines, camels, heep, pigs, or rats) it is routine work forthe skilled person to make a variant of such a parent chymosin ofinterest when guided by present disclosure.

An example of a suitable method to produce and isolate a chymosin(variant or parent) may be by well-known e.g. fungal recombinantexpression/production based technology as e.g. described in WO02/36752A2(Chr. Hansen).

It is also routine work for the skilled person to make alteration at oneor more positions in a parent polypeptide having chymosin activity,wherein the alteration is comprising a substitution, a deletion or aninsertion in at least one amino acid position as disclosed herein.

As known to the skilled person—this may e.g. be done by so-called sitedirected mutagenesis and recombinant expression/production basedtechnology.

It is also routine work for the skilled person to determine if a hereinrelevant parent polypeptide (e.g. camel or bovine wildtype chymosin)and/or a herein relevant variant has chymosin activity or not.

As known in the art—chymosin specificity may be determined by theso-called C/P ratio, which is determined by dividing the specificclotting activity (C) with the proteolytic activity (P). As known in theart—a higher C/P ratio implies generally that the loss of protein duringe.g. cheese manufacturing due to non-specific protein degradation isreduced, i.e. the yield of cheese is improved.

Determination of Milk Clotting Activity

Milk clotting activity may be determined using the REMCAT method, whichis the standard method developed by the International Dairy Federation(IDF method). Milk clotting activity is determined from the time neededfor a visible flocculation of a standard milk substrate prepared from alow-heat, low fat milk powder with a calcium chloride solution of 0.5 gper liter (pH≈6.5). The clotting time of a rennet sample is compared tothat of a reference standard having known milk-clotting activity andhaving the same enzyme composition by IDF Standard 110B as the sample.Samples and reference standards are measured under identical chemicaland physical conditions. Variant samples are adjusted to approximately 3IMCU/ml using an 84 mM acetic acid buffer pH 5.5. Hereafter, 20 μlenzyme preparation is added to 1 ml preheated milk (32° C.) in a glasstest tube placed in a water bath, capable of maintaining a constanttemperature of 32° C.±1° C. under constant stirring.

The total milk-clotting activity (strength) of a rennet is calculated inInternational Milk-Clotting Units (IMCU) per ml relative to a standardhaving the same enzyme composition as the sample according to theformula:

${{Strength}\mspace{14mu}{in}\mspace{14mu}{{IMCU}/{ml}}} = \frac{{Sstandard} \times {Tstandard} \times {Dsample}}{{Dstandard} \times {Tsample}}$

-   -   Sstandard: The milk-clotting activity of the international        reference standard for rennet.    -   Tstandard: Clotting time in seconds obtained for the standard        dilution.    -   Dsample: Dilution factor for the sample    -   Dstandard: Dilution factor for the standard    -   Tsample: Clotting time in seconds obtained for the diluted        rennet sample from addition of enzyme to time of flocculation.

For clotting activity determination the μIMCU method may be used insteadof the REMCAT method. As compared to REMCAT, flocculation time ofchymosin variants in the μIMCU assay is determined by OD measurements in96-well microtiter plates at 800 nm in a UV/VIS plate reader. A standardcurve of various dilutions of a reference standard with known clottingstrength is recorded on each plate. Samples are prepared by dilutingenzyme in 84 mM acetate buffer, 0.1% triton X-100, pH 5.5. Reaction at32° C. is started by adding 250 uL of a standard milk substratecontaining 4% (w/w) low-heat, low fat milk powder and 7.5% (w/w) calciumchloride (pH 6.5) to 25 uL enzyme sample. Milk clotting activity ofchymosin variants in International Milk-Clotting Units (IMCU) per ml isdetermined based on sample flocculation time relative to the standardcurve.

Determination of Total Protein Content

Total protein content may preferably be determined using the Pierce BCAProtein Assay Kit from Thermo Scientific following the instructions ofthe providers.

Calculation of Specific Clotting Activity

Specific clotting activity (IMCU/mg total protein) was determined bydividing the clotting activity (IMCU/ml) by the total protein content(mg total protein per ml).

Determination of Proteolytic Activity

General proteolytic activity may preferably be measured usingfluorescently labelled Bodipy-FL casein as a substrate (EnzChek;Molecular Bioprobes, E6638). Casein derivatives heavily labeled withpH-insensitive green-fluorescent Bodipy-FL result in quenching of theconjugate's fluorescence. Protease catalyzed hydrolysis releasesfluorescent Bodipy-FL. This method is very sensitive which was essentialfor this experiment as the reference has the lowest generalproteolytical activity of all coagulants known to date. A 0.04 mg/mlsubstrate solution is prepared in 0.2M phosphate buffer pH 6.5,containing 100 mM NaCI, 5% glycerol, and 0.1% Brij. Chymosin variantsare dissolved in 20 mM malonate buffer, containing 100 mM NaCl, 5%glycerol, and 0.1% Brij. Of both reference and chymosin variantsolutions, 20 μL are mixed in a black 384-well Corning flat bottompolystyrene microtitter plate and fluorescence was continuously recordedin a fluorometer at 32C for 10 hours. Slopes of the linear part offluorescence change are used to determine general proteolytic activity.

Determination of the C/P Ratio

The C/P ratio is calculated by dividing the clotting activity (C) withthe proteolytic activity (P).

Statistical Analysis of the Positional and Mutational Effects onSpecific Clotting Activity and C/P Ratio

A statistical machine-learning approach and PCA-based analysis maypreferably be used to determine the effects of single mutations presentin the multi-substitution variants, i.e. specific milk clottingactivity, as well as on the ratio of clotting and general proteolyticactivity (C/P).

Preferred Embodiments of the Invention

As outlined above and illustrated in the examples below, the inventorsof present disclosure have made a number of preferred chymosinpolypeptide variants with improved clotting activity and/or C/P ratiowhen compared to the corresponding parent polypeptide under comparableconditions.

In a preferred aspect, the present invention relates to an isolatedchymosin polypeptide variant comprising an alteration in one or morepositions compared to a parent polypeptide having chymosin activity,wherein the alteration comprise a substitution, a deletion or aninsertion in at least one amino acid position corresponding to any ofpositions: D59, V309, S132, N249, L166, N249, Q56, Y134, K295, M157,M256, R242, I96, H76, S164, S273, G251, Y11, L166, K19, Y21, S74, Y243,N249, S273, Q280, F282, L295, N252, R254, G70, V136, L222, K231, N291.

wherein

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO:2 and

(ii): the parent polypeptide has at least 80% sequence identity with themature polypeptide of SEQ ID NO:2 (camel chymosin);

wherein the isolated chymosin polypeptide variant has a higher specificclotting activity and/or C/P ratio than its corresponding parentpolypeptide.

More specifically, the isolated chymosin polypeptide variant of presentinvention has a specific clotting activity (IMCU/mg total protein) thatis at least 85% such as e.g. at least 90%, 100%, 110%, 120%, 130%, 160%or 200% of the specific clotting activity of its parent polypeptide.Alternatively or additionally, the isolated chymosin polypeptide variantof present invention has a C/P ratio that is at least 200%, such as e.g.at least 400%, at least 500%, at least 750% or at least 1000% of the C/Pratio of its parent polypeptide.

As specified above, the parent polypeptide may have at least 80%, suchas at least e.g. 82%, 85%, 95%, 97%, 98%, 99% or 100% sequence identitywith the mature polypeptide of SEQ ID NO:2 (camel chymosin).

The alteration may comprise a substitution selected from a listconsisting of:

-   D59N, V309I, S132A, N249E, L166V, N249D, Q56H, Y134G, K295L, M157L,    M256L, R242E, I96L, H76Q, S164G, S273Y, G251D, Y11I, R242D, L222V,    Y11V, L166I, K19T, Y21S, S74D, Y243E, N249D, S273D, Q280E, F282E,    L295K, N252D, R254E, G70D, V136I, L222I, K231N, N291Q.

In a related embodiment, the present invention relates to an isolatedchymosin polypeptide variant wherein the alteration comprises one ormore combinations of substitutions comprising:

-   Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I;-   Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I;-   Y11I, I96L, S164G, L222I, R242E;-   Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D;-   H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;-   K19T, D59N, H76Q, S164G, L222I, N249D, S273Y;-   K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y;-   K19T, D59N, H76Q, S132A, L222I, G251D, S273Y, V309I;-   Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y;-   D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y;-   D59N, H76Q, I96L, S132A, S164G, L166V, L222I, G251D, S273Y;-   H76Q, S164G, L166V, L222I, R242E, G251D, S273Y;-   D59N, H76Q, S132A, S164G, L166V, S273Y;-   K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y;-   Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y;-   K19T, D59N, I96L, S164G, L222I, G251D;-   D59N, H76Q, S164G, L222I, S226T, R242E;-   H76Q, L130I, L222I, S226T, G251D, S273Y;-   Y21S, D59N, H76Q, I96L, L222I, S273Y;-   H76Q, S164G, L222I, N249D, G251D, S273Y, V309I;-   D59N, I96L, L166V, L222I, R242E, G251D;-   Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I;-   K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D;-   K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D;-   H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;-   K19T, I96L, L222I, R242E, L253I;-   K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I;-   I96L, S164G, L222I, R242E, G251D, S274Y;-   R242E, N252D, N100Q, N291Q;-   R242E, R254E, Q280E, N100Q, N291Q;-   R242E, Q280E, N100Q, N291Q;-   R242E, R254E, S273D, Q280E, N100Q, N291Q;-   R67Q, S132A, L222I, K231N, R242E, V248I;-   R67Q, I96L, L130I, M157L, K231N, R242E;-   R67Q, M157L, L222I, K231N, V248I;-   R67Q, I96L, M157L, L222I, K231N or-   R67Q, G70D, M157L, L222I, N291Q;

Optionally, the isolated chymosin polypeptide variant may furthercomprise substitutions that alter the glycosylation pattern, such ase.g. substitutions in one or more of positions N100, N252 and/or N291,more specifically N100Q, N252Q and/or N291Q.

Preferred Methods for Making Isolated Chymosin Polypeptide Variants

The present invention further relates to methods for producing anisolated polypeptide according to present disclosure.

As a related embodiment, the present invention relates to a method formaking an isolated chymosin polypeptide variant wherein the methodcomprises the steps:

(a): making an alteration at one or more positions in a parentpolypeptide, wherein the alteration is comprising a substitution, adeletion or an insertion in at least one amino acid positioncorresponding to any of positions: D59, V309, S132, N249, L166, N249,Q56, Y134, K295, M157, M256, R242, I96, H76, S164, S273, G251, Y11,L166, K19, Y21, S74, Y243, N249, S273, Q280, F282, L295, N252, R254,G70, V136, L222, K231, N291,

(b): producing and isolating the altered polypeptide of step (a), andwherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO:2 (camel chymosin); and

(ii): the parent polypeptide has at least 80% sequence identity with themature polypeptide of SEQ ID NO:3 (bovine chymosin) and/or at least 80%sequence identity with the mature polypeptide of SEQ ID NO:2 (camelchymosin).

More specifically, the alteration may be one or more of thesubstitutions: D59N, V309I, S132A, N249E, L166V, N249D, Q56H, Y134G,K295L, M157L, M256L, R242E, I96L, H76Q, S164G, S273Y, G251D, Y11I,R242D, L222V, Y11V, L166I, K19T, Y21S, S74D, Y243E, N249D, S273D, Q280E,F282E, L295K, N252D, R254E, G70D, V136I, L222I, K231N, N291Q.

In yet a related aspect, the present invention relates to a method formaking an isolated chymosin polypeptide variant as specified above,wherein the isolated chymosin polypeptide variant comprises substitutionin one or more of the combinations of positions comprising the positionscorresponding to:

-   Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I;-   Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I;-   Y11I, I96L, S164G, L222I, R242E;-   Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D;-   H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;-   K19T, D59N, H76Q, S164G, L222I, N249D, S273Y;-   K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y;-   K19T, D59N, H76Q, S132A, L222I, G251D, S273Y, V309I;-   Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y;-   D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y;-   D59N, H76Q, I96L, S132A, S164G, L166V, L222I, G251D, S273Y;-   H76Q, S164G, L166V, L222I, R242E, G251D, S273Y;-   D59N, H76Q, S132A, S164G, L166V, S273Y;-   K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y;-   Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y;-   K19T, D59N, I96L, S164G, L222I, G251D;-   D59N, H76Q, S164G, L222I, S226T, R242E;-   H76Q, L130I, L222I, S226T, G251D, S273Y;-   Y21S, D59N, H76Q, I96L, L222I, S273Y;-   H76Q, S164G, L222I, N249D, G251D, S273Y, V309I;-   D59N, I96L, L166V, L222I, R242E, G251D;-   Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I;-   K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D;-   K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D;-   H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;-   K19T, I96L, L222I, R242E, L253I;-   K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I;-   I96L, S164G, L222I, R242E, G251D, S274Y;-   R242E, N252D, N100Q, N291Q;-   R242E, R254E, Q280E, N100Q, N291Q;-   R242E, Q280E, N100Q, N291Q;-   R242E, R254E, S273D, Q280E, N100Q, N291Q;-   R67Q, S132A, L222I, K231N, R242E, V248I;-   R67Q, I96L, L130I, M157L, K231N, R242E;-   R67Q, M157L, L222I, K231N, V248I;-   R67Q, I96L, M157L, L222I, K231N or-   R67Q, G70D, M157L, L222I, N291Q.

The parent polypeptide may as a preferred embodiment have at least 95%sequence identity with the mature polypeptide of SEQ ID NO:2 (Camelchymosin).

Further the present invention relates to method for making a food orfeed product comprising adding an effective amount of the isolatedchymosin polypeptide variant as described herein to the food or feedingredient(s) and carrying our further manufacturing steps to obtain thefood or feed product.

A further related aspect of present invention concerns a method formaking a food or feed product comprising adding an effective amount ofthe isolated chymosin polypeptide variant as described herein to thefood or feed ingredient(s) and carrying our further manufacturing stepsto obtain the food or feed product, in particular wherein the food orfeed product is a milk-based product or a food or feed productcomprising a chymosin polypetide of present invention.

A further related aspect of present invention relates to a chymosinpolypetide variant according to present invention in a process formaking a milk based product such as e.g. cheese, such as e.g. pastafilata, cheddar, continental type cheeses, soft cheese or white brinecheese.

As discussed above—an isolated chymosin polypeptide variant as describedherein may be used according to the art—e.g. to make a milk basedproduct of interest (such as e.g. a cheese product).

As discussed above—an aspect of the invention relates to a method formaking a food or feed product comprising adding an effective amount ofthe isolated chymosin polypeptide variant as described herein to thefood or feed ingredient(s) and carrying our further manufacturing stepsto obtain the food or feed product.

Preferably, the food or feed product is a milk-based product and whereinthe method comprises adding an effective amount of the isolated chymosinpolypeptide variant as described herein to milk and carrying our furthermanufacturing steps to obtain the milk based product.

The milk may e.g. be soy milk, sheep milk, goat milk, buffalo milk, yakmilk, lama milk, camel milk or cow milk.

The milk based product may e.g. be a fermented milk product such as aquark or a cheese.

As known in the art, the growth, purification, testing and generalhandling may influence the performance of enzymes and hence also theenzyme of present invention. Hence the present invention relates tochymosin polypeptide variants, methods for making these and productscontaining these, wherein the chymosin polypeptide variant has animproved clotting activity and/or C/P ratio when compared to thecorresponding parent polypeptide under comparable conditions andpreferably after being produced and otherwise handled under comparableconditions.

EXAMPLES Example 1 Alignment and Numbering of Chymosin Protein Sequencesand Variant Sequences

Chymosin protein sequences were aligned using the ClustalW algorithm asprovided by the EBI (EBI, tools, multiple sequence alignment, CLUSTALW″,http://www.ebi.ac.uk/Tools/msa/clustalw2/) and as described in Larkin MA, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H,Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J,Higgins D G (2007). Bioinformatics 23(21), 2947-2948.

ClustalW2 settings for multiple sequence alignments were Protein weightMatrix=BLOSUM, GAP open=10, GAP EXTENSION=0.5, GAP DISTANCES=8, No EndGaps, ITERATION=none, NUMITER=1, CLUSTERING=NJ

As a reference sequence the bovine chymosin B preprochymosin was used(Gen-bank accession number P00794—disclosed herein as SEQ ID NO:1),where the N-terminal Methionin has number 1 (MRCL . . . ) and theC-terminal Isoleucin (in the protein sequence . . . LAKAI) has number381. Variants were aligned against the bovine B pre-pro-chymosin andresidues were numbered according to the corresponding bovine chymosinresidue.

Example 2 Design of Chymosin Variants

Chymosin variants were designed using different strategies.

When there is referred to camel chymosin there is referred to camelchymosin comprising the polypeptide of SEQ ID NO:2 herein.

Camel chymosin of SEQ ID NO:2 may be seen as a herein relevant parentpolypeptide having chymosin activity used to make camel chymosinvariants thereof.

When there is referred to bovine chymosin there is referred to bovinechymosin comprising the polypeptide of SEQ ID NO:1 herein. Bovinechymosin of SEQ ID NO:1 may be seen as a relevant parent polypeptidehaving chymosin activity used to make bovine chymosin variants thereof.

Variants 180 to 269 and 367 to 461 of camel chymosin were designed basedon an alignment of a large set of public known aspartic proteasesequences having an identity of 25% or more compared to bovine chymosinB.

Variations were generally introduced in regions with a high level ofamino acid variation between species, while conserved regions were notchanged. Amino acid substitutions were chosen based on phylogenetic,structural and experimental information to identify changes with highprobability to show beneficial effects on specific clotting activity andthe C/P ratio. Multiple variations were introduced in each variantconstruct, ensuring that each single mutation was present in multiplevariant constructs to minimize the effect of covariation between varioussubstitutions. Machine learning and statistical analysis of experimentaldata were used to determine the relative contributions of the amino acidsubstitutions to measured coagulant performance of the chymosin variants(references 14, 15).

Variants 271 to 366 were designed based on detailed structural analysisof bovine chymosin (PDB code: 4AA8) and camel chymosin (PDB code: 4AA9).Variations were chosen based on the chemical nature of the respectiveamino acid side chains and their expected impact on either caseinsubstrate binding or general enzyme properties. Most of the amino acidsubstitutions in variants 271 to 346 were made in sequence positionseither within or in close structural proximity to the substrate bindingcleft, or in secondary structural elements that get into contact withthe bound casein substrate. Furthermore, changes were made in positionson the protein surface that alter the charge profile of these regions(reference 5) and are therefore expected to have an impact on enzymeperformance. Variants 347 to 366 were made based on the differentstructural conformation of the N-terminal sequence in bovine and camelchymosin. Amino acid substitutions were made in positions within thesubstrate binding cleft that interact with the N-terminus in camelchymosin.

Example 3 Preparation of Chymosin Variant Enzyme Material

All chymosin variants were synthesized as synthetic genes and clonedinto a fungal expression vector such as e.g. pGAMpR-C (described inWO02/36752A2) The vectors were transformed into E. coli and plasmid DNAwas purified using standard molecular biology protocols, known to theperson skilled in the art. The variant plasmids were individuallytransformed into an Aspergillus niger or Aspergillus nidulans strain andprotein was produced essentially as described in WO02/36752A2 andpurified using standard chromatography techniques. For enzyme libraryscreening, all chymosin variants were produced in 20 -60 mLfermentations. For more detailed characterization of variants 433, 436,453, and 457, the respective enzymes were fermented again in 70L scale.

As known in the art—the skilled person may, based on his common generalknowledge, produce and purify chymosin and chymosin variants—such asherein described bovine and camel chymosin variants.

Example 4 Determination of Specific Chymosin Activity 4.1 Determinationof Milk Clotting Activity

Milk clotting activity was determined using the REMCAT method, which isthe standard method developed by the International Dairy Federation (IDFmethod). Milk clotting activity is determined from the time needed for avisible flocculation of a standard milk substrate prepared from alow-heat, low fat milk powder with a calcium chloride solution of 0.5 gper liter (pH≈6.5). The clotting time of a rennet sample is compared tothat of a reference standard having known milk-clotting activity andhaving the same enzyme composition by IDF Standard 110B as the sample.Samples and reference standards were measured under identical chemicaland physical conditions. Variant samples were adjusted to approximately3 IMCU/ml using an 84 mM acetic acid buffer pH 5.5. Hereafter, 20 μlenzyme preparation was added to 1 ml preheated milk (32° C.) in a glasstest tube placed in a water bath, capable of maintaining a constanttemperature of 32° C.±1° C. under constant stirring.

The total milk-clotting activity (strength) of a rennet was calculatedin International Milk-Clotting Units (IMCU) per ml relative to astandard having the same enzyme composition as the sample according tothe formula:

${{Strength}\mspace{14mu}{in}\mspace{14mu}{{IMCU}/{ml}}} = \frac{{Sstandard} \times {Tstandard} \times {Dsample}}{{Dstandard} \times {Tsample}}$

-   Sstandard: The milk-clotting activity of the international reference    standard for rennet.-   Tstandard: Clotting time in seconds obtained for the standard    dilution.-   Dsample: Dilution factor for the sample-   Dstandard: Dilution factor for the standard-   Tsample: Clotting time in seconds obtained for the diluted rennet    sample from addition of enzyme to time of flocculation.

For clotting activity determination of libraries 1 and 3 variants aswell as variants by structural design, the μIMCU method was used insteadof the REMCAT method. As compared to REMCAT, flocculation time ofchymosin variants in the μIMCU assay was determined by OD measurementsin 96-well microtiter plates at 800 nm in a UV/VIS plate reader. Astandard curve of various dilutions of a reference standard with knownclotting strength was recorded on each plate. Samples were prepared bydiluting enzyme in 84 mM acetate buffer, 0.1% triton X-100, pH 5.5.Reaction at 32° C. was started by adding 250 uL of a standard milksubstrate containing 4% (w/w) low-heat, low fat milk powder and 7.5%(w/w) calcium chloride (pH≈6.5) to 25 uL enzyme sample. Milk clottingactivity of chymosin variants in International Milk-Clotting Units(IMCU) per ml was determined based on sample flocculation time relativeto the standard curve.

4.2 Determination of Total Protein Content

Total protein content was determined using the Pierce BCA Protein AssayKit from Thermo Scientific following the instructions of the providers.

4.3 Calculation of Specific Clotting Activity

Specific clotting activity (IMCU/mg total protein) was determined bydividing the clotting activity (IMCU/ml) by the total protein content(mg total protein per ml).

Example 5 Determination of Proteolytic Activity

General proteolytic activity was measured using fluorescently labelledBodipy-FL casein as a substrate (EnzChek; Molecular Bioprobes, E6638).Casein derivatives heavily labeled with pH-insensitive green-fluorescentBodipy-FL result in quenching of the conjugate's fluorescence. Proteasecatalyzed hydrolysis releases fluorescent Bodipy-FL. This method is verysensitive which was essential for this experiment as CHYMAX M has thelowest general proteolytical activity of all coagulants known to date.

A 0.04 mg/ml substrate solution was prepared in 0.2M phosphate buffer pH6.5, containing 100 mM NaCI, 5% glycerol, and 0.1% Brij. Chymosinvariants were solved in 20 mM malonate buffer, containing 100 mM NaCI,5% glycerol, and 0.1% Brij. Of both substrate and chymosin variantsolutions, 20 μL were mixed in a black 384-well Corning flat bottompolystyrene microtitter plate and fluorescence was continuously recordedin a fluorometer at 32° C. for 10 hours. Slopes of the linear part offluorescence change were used to determine general proteolytic activity.

Example 6 Statistical Analysis of the Positional and Mutational Effectson Specific Clotting Activity and C/P Ratio

A statistical machine-learning approach and PCA-based analysis was usedto determine the effects of all single mutations present in the variantsof multisubstitution libraries 1 to 3 on cleavage of κ-casein betweenpositions Phe105 and Met106, i.e. specific milk clotting activity, aswell as on the ratio of clotting and general proteolytic activity (C/P).

Results

Multi-Substitution Library 1

Variants of camel chymosin, each having multiple substitutions comparedto wild type, were generated and analyzed as described above. Allvariants have an amino acid sequence identical to camel chymosin (SEQ IDNO:2), except for the variations mentioned in the table. Camel chymosin(CHY-MAX M) is included as reference.

Clotting activities were determined using the pIMCU method.

TABLE 1 Enzymatic activities of camel chymosin variants 180-222. Numbersare given in % cleavage of wild type camel chymosin (CHY-MAX M).Clotting Proteolytic variant (C) (P) C/P CHY-MAX M mutations 100 100 100180 H76Q S132A S164G L222I N249D G251D 72 37 194 181 Y21S D59N H76QS164G L166V N249D G251D S273Y 77 37 210 182 D59N H76Q S164G L222I R242ES273Y V309I 96 21 449 183 D59N H76Q L130I L166V L222I N249D G251D S273Y84 55 152 184 Y21S D59N S164G L222I R242E G251D S273Y V309I 102 35 287185 K19T Y21S D59N H76Q S132A S164G L222I G251D S273Y 97 29 334 186 D59NH76Q I96L L130I S164G L222I R242E G251D 85 16 524 187 H76Q S164G L166VL222I S226T S273Y 103 21 504 188 K19T D59N I96L S164G L222I G251D 126 31403 189 Y21S H76Q S164G L222I R242E G251D S273Y 138 14 975 190 H76Q I96LS164G L222I R242E G251D S273Y 153 10 1479 191 H76Q S164G L222I N249DG251D S273Y V309I 112 19 606 192 K19T D59N H76Q S164G L222I N249D S273Y152 42 363 193 Y21S D59N H76Q S164G L222I S226T G251D S273Y V309I 107 32340 194 H76Q S164G L166V L222I R242E G251D S273Y 132 14 949 195 D59NH76Q I96L S164G L222I S226T N249D G251D S273Y 96 19 498 196 D59N H76QL130I S164G L166V L222I G251D S273Y V309I 76 24 316 197 D59N S132A S164GL222I R242E N249D G251D S273Y 138 38 365 198 H76Q I96L S164G G251D S273YV309I 71 16 443 199 D59N H76Q L130I S164G G251D V309I 54 18 309 200 K19TD59N S164G L166V L222I S226T G251D S273Y 107 31 342 201 D59N H76Q I96LS132A S164G L222I S226T G251D S273Y 96 23 426 202 K19T D59N H76Q I96LS164G L166V L222I G251D S273Y 90 41 218 203 K19T D59N H76Q L130I S164GL222I S226T G251D S273Y 64 21 309 204 K19T D59N H76Q S132A L222I G251DS273Y V309I 141 48 294 205 H76Q L130I L222I S226T G251D S273Y 124 38 322206 K19T Y21S D59N H76Q L130I S164G L222I S273Y 75 25 295 207 Y21S D59NH76Q I96L S164G L222I N249D G251D S273Y 129 17 762 208 K19T D59N H76QS164G R242E N249D G251D S273Y 129 15 879 209 D59N H76Q S164G L222I S226TR242E 124 30 417 210 D59N H76Q I96L S132A S164G L166V L222I G251D S273Y136 21 657 211 D59N H76Q S132A S164G L166V S273Y 131 31 423 212 Y21SD59N S164G L222I S226T N249D G251D S273Y 92 48 190 213 D59N H76Q L130IS132A S164G L222I R242E G251D S273Y 108 24 441 214 D59N H76Q S164G L166VL222I N249D G251D S273Y V309I 111 65 171 215 D59N H76Q I96L S164G L222IS226T G251D S273Y V309I 87 24 369 216 K19T D59N H76Q L166V L222I R242EG251D S273Y 146 30 494 217 Y21S D59N H76Q I96L L222I S273Y 118 52 228218 D59N H76Q I96L L130I S164G L222I N249D G251D S273Y 75 23 323 219L130I S164G L222I S273Y 46 38 121 220 K19T Y21S H76Q S164G L222I G251DS273Y 65 28 228 221 Y21S D59N H76Q L130I S132A S164G L222I G251D S273Y65 31 213 222 D59N H76Q S226T R242E G251D S273Y 102 37 273

In table 1 are shown camel chymosin variants with data on specificclotting activity (C), unspecific proteolytic activity (P) as well asthe C/P ratio. Out of 43 variants 17 reveal between 10% and 50%increased specific clotting activity compared to wild type camelchymosin (CHY-MAX M). All variants have significantly increased C/Pratios, with the best one, 190, showing a ca. 15× improvement comparedto wild type camel chymosin.

Mutational Analysis of Multi-Substitution Library 1

A statistical analysis of the positional and mutational effects onspecific clotting activity (C) and the C/P ratio was performed based onthe proteolytic data of library 1. The most beneficial mutations forincreased specific clotting and C/P are shown in tables 2 and 3,respectively.

TABLE 2 Mutational contributions (mean) to increased specific clottingactivity and standard deviations (sd) based on statistical analysis.mutation mean sd R242E 1.98E−01 2.47E−02 L222I 1.09E−01 3.35E−02 D59N6.06E−02 3.12E−02 S273Y 6.06E−02 3.47E−02 K19T 5.13E−02 2.65E−02 V309I4.37E−02 2.92E−02 S132A 4.18E−02 2.46E−02 N249D 3.85E−02 2.54E−02 I96L3.38E−02 2.59E−02

Based on the results shown in table 2 it is concluded that mutationsK19T, 1359N, I96L, S132A, L222I, R242E, N249D, S273Y, and V309I increasethe specific clotting activity of chymosin. It can consequently beexpected that these mutations enable a lower dosing of chymosin incheese manufacturing.

TABLE 3 Mutational contributions (mean) to increased C/P ratio andstandard deviations (sd) based on statistical analysis. mutation mean sdR242E 2.12E−01 2.82E−02 I96L 1.20E−01 2.81E−02 H76Q 9.10E−02 2.16E−02S164G 8.59E−02 2.19E−02 S273Y 7.77E−02 2.01E−02 G251D 3.59E−02 1.99E−02

Based on the results shown in table 3 it is concluded that mutationsH76Q, I96L, S164G, R242E, G251D, and S273Y increase the C/P ratio ofchymosin. It can consequently be expected that these mutations result inincreased yields during cheese manufacturing using the respectivechymosin variants.

Multi-Substitution Library 2

Another set of camel chymosin variants, each having multiplesubstitutions compared to wild type, were generated and analyzed asdescribed above. All variants have an amino acid sequence identical tocamel chymosin (SEQ ID NO:2), except for the variations mentioned in thetable. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the REMCAT method.

TABLE 4 Enzymatic activities of camel chymosin variants 223-269. Numbersare given in % cleavage of wild type camel chymosin (CHY-MAX M).Clotting Proteolytic variant (C) (P) C/P CHY-MAX M mutations 100 100 100223 K19T D59N I96L S164G L222I G251D 89 37 242 224 Y11I K19T D59N I96VL222I R242D G251D 82 31 262 225 K19S D59N I96V S164G G251D 72 40 182 226K19S I96L S164G L166V L222I R242E 91 38 242 227 K19T D59N I96L S164GL166V L222I R242D G251D L253I 92 24 378 228 D59N I96L S164G L222I R242EL253I I263L 108 23 467 229 K19T D59N E83T I96L L222I G251D I263L 99 10693 230 Y11I K19T D59N S164G L222I G251D I263V 54 16 343 231 K19T D59NI96L S164G L166I G251D L253V 63 30 206 232 K19T I96V S164G L222I N249DG251D L253I 56 29 193 233 K19T I96L L222I R242E L253I 125 57 220 234K19T E83S I96L S164G L222I R242E G251D L253I 83 35 235 235 D59N E83TI96L S164N L222V G251D 42 53 80 236 K19S D59N I96L S164G L222I R242EN249E G251D 130 28 459 237 K19T I96L S164G L166V L222I N249D I263L 65 30217 238 D59N I96L L166V L222I R242E G251D 178 51 347 239 K19T D59N E83TS164G L166V L222I R242D G251D 101 43 235 240 Y11I K19T D59N E83S I96LS164G L222I N249D 53 60 87 241 K19T E83T I96L S164G L222I R242E L253V 9737 261 242 K19T D59N I96L S164G L166I L222I R242E N249D 129 21 623 243Y11V K19T D59N I96L S164G L166V L222I R242E G251D L253I 130 17 759 244K19T I96L S164N L222I R242E I263L 51 22 236 245 Y11V D59N I96L S164GL222I G251D L253V 63 24 265 246 K19T D59N I96V S164G L166V L222I R242EI263L 98 28 347 247 Y11V K19T D59N I96L S164N L166I L222I G251D 32 16202 248 K19T I96L S164G L166V L222I R242E N249D G251D I263V 105 19 566249 K19T I96L S164G R242E L253I 73 14 516 250 K19S D59N E83S I96L S164NL222I G251D 47 64 74 251 K19T D59N I96L S164G L222V N249E G251D I263V 7927 293 252 K19T D59N I96L S164G L222I N249E G251D L253V I263L 69 21 332253 Y11I K19T I96L S164G L222V R242E G251D 58 2 3265 254 I96L S164GL222I R242E N249D G251D I263L 82 14 601 255 K19T D59N I96L S164G L166IL222I R242D G251D I263V 108 25 427 256 K19T D59N I96L S164G L222V R242EN249D L253I 111 19 574 257 H76Q I96L S164G L222I R242E G251D S273Y 128 81597 258 K19T E83S I96L S164G L222I R242E N249D G251D L253I 95 30 315259 I96L S164G L166V L222I R242E N249D I263L 104 26 405 260 Y11V K19TE83S I96L S164G L166V L222I R242E G251D 97 14 676 261 Y11V K19T I96LS164G L166V L222I R242E 94 19 491 262 Y11V E83S I96L S164G L222I R242EG251D L253I I263L 61 18 332 263 Y11V I96L S164G L222I R242E N249D L253II263L 67 7 961 264 K19T I96L S164G L166V L222I R242E N249D I263L 75 50149 265 Y11V E83S I96L S164G L222I R242E L253I I263L 62 28 222 266 K19TE83S I96L S164G L166V L222I R242E N249D G251D L253I 97 32 302 267 I96LS164G L222I R242E G251D S274Y 110 19 569 268 H76Q I96L S164G L222I R242EG251D 102 10 1054 269 I96L S164G L222I R242E G251D 101 22 465

In table 4 are shown camel chymosin variants with data on specificclotting activity (C), unspecific proteolytic activity (P) as well asthe C/P ratio. Out of 47 variants, 8 reveal between 10% and 78%increased specific clotting activity compared to wild type camelchymosin (CHY-MAX M). While 43 variants have significantly increased C/Pratios, the best one, 253, shows a ca. 33× improvement compared to wildtype camel chymosin.

Mutational Analysis of Multi-Substitution Library 2

A statistical analysis of the positional and mutational effects onspecific clotting activity (C) and the C/P ratio was performed based onthe proteolytic data of library 2. The most beneficial mutations forincreased specific clotting and C/P are shown in tables 5 and 6,respectively.

TABLE 5 Mutational contributions (mean) to increased specific clottingactivity and standard deviations (sd) based on statistical analysis.mutation mean sd R242E 4.00E−01 3.19E−02 D59N 2.94E−01 2.26E−02 N249E1.47E−01 3.22E−02 L166V 1.27E−01 2.70E−02 S273Y 1.23E−01 2.94E−02 L222I1.07E−01 3.53E−02 H76Q 5.93E−02 2.94E−02 N249D 4.26E−02 2.38E−02

Based on the results shown in table 5 it is concluded that mutationsD59N, H76Q, L166V, L222I, R242E, N249D, N249E, and S273Y increase thespecific clotting activity of chymosin. It can consequently be expectedthat these mutations enable a lower dosing of chymosin in cheesemanufacturing.

TABLE 6 Mutational contributions (mean) to increased C/P ratio andstandard deviations (sd) based on statistical analysis. mutation mean sdR242E 4.13E−01 2.20E−02 H76Q 2.50E−01 3.24E−02 Y11I 2.49E−01 6.43E−02S164G 2.27E−01 2.07E−02 G251D 2.10E−01 2.65E−02 R242D 1.85E−01 2.69E−02L222V 1.75E−01 4.53E−02 Y11V 1.75E−01 2.83E−02 S273Y 8.29E−02 3.35E−02L166I 7.64E−02 2.91E−02 I96L 3.85E−02 2.59E−02 K19T 3.85E−02 2.43E−02

Based on the results shown in table 6 it is concluded that mutationsY11I, Y11V, K19T, H76Q, I96L, S164G, L166I, L222V, R242D, R242E, G251D,and S273Y increase the C/P ratio of chymosin. It can consequently beexpected that these mutations result in increased yields during cheesemanufacturing using the respective chymosin variants.

Structure-Based Variations in Camel Chymosin

Variants of camel chymosin (SEQ ID NO:2) were made with amino acidchanges in positions determined by protein structural analysis (Tab. 7).Mutations N100Q and N291Q were introduced into both N-glycosylationsites of these variants and the reference camel chymosin (CamUGly) toyield non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the pIMCU method.

TABLE 7 Enzymatic activities of camel chymosin variants 271-308. Numbersare given in % cleavage of non-glycosylated camel chymosin (CamUGly).Table 7 CamBov Clotting Proteolytic variant mutations (C) (P) C/PCamUGly N100Q N291Q 100 100 100 271 V221K N100Q N291Q 47 61 77 272 D290EN100Q N291Q 92 100 92 273 V136I N100Q N291Q 80 90 89 274 E240Q N100QN291Q 84 144 58 276 G289S N100Q N291Q 93 107 86 277 N292H N100Q N291Q 9593 100 278 L295K N100Q N291Q 102 70 146 279 V136E N100Q N291Q 102 102100 280 D290L N100Q N291Q 44 198 22 281 F119Y N100Q N291Q 8 45 18 282Q280E N100Q N291Q 79 72 110 283 F282E N100Q N291Q 93 80 116 284 N249DN100Q N291Q 118 84 140 285 R254S N100Q N291Q 47 94 50 286 R242E N100QN291Q 114 67 170 288 V203R N100Q N291Q 99 113 88 289 N249R N100Q N291Q76 108 70 290 H56K N100Q N291Q 99 133 74 291 S74D N100Q N291Q 94 87 108292 A131D N100Q N291Q 17 39 44 293 Y190A N100Q N291Q 3 33 9 294 I297AN100Q N291Q 26 37 70 302 Y21S N100Q N291Q 97 87 111 303 L130I N100QN291Q 77 82 95 306 G251D N100Q N291Q 100 81 123 307 Y243E N100Q N291Q 8658 149 308 S273D N100Q N291Q 102 98 104

Based on the results shown in table 7 it is concluded that mutationsY21S, S74D, R242E, Y243E, N249D, G251D, S273D, Q280E, F282E, and L295Kincrease the C/P ratio of chymosin. Mutations R242E and N249D alsoresult in increased specific clotting activity. Seven out of tenvariants with increased C/P ratios shown in table 7 bear mutations(R242E, N249D, G251D, Y243E, S273D, Q280E, F282E) in a distinct regionon the protein surface that is located in proximity to the binding cleftas seen in FIG. 2. This region has been suggested to support binding ofthe κ-casein substrate by interacting with its positively chargedsequence Arg96 to His102 (references 5, 16-18) in positions P10 to P4(reference 10). The negative charges introduced with the mutations maystrengthen these interactions, resulting in increased specificity forκ-casein (C/P). The results show that single amino acid substitutions inthis region can increase C/P significantly.

Negative Charge Combinations in Camel Chymosin

More variants of camel chymosin (SEQ ID NO:2) were made withcombinations of mutations that introduce negative charges into thesurface region described above (R242E, Y243E, G251D, N252D, R254E,S273D, Q280E). Mutations N100Q and N291Q were introduced into bothN-glycosylation sites of these variants and the reference camel chymosin(CamUGly) to yield non-glycosylated, homogeneous protein samples (Tab.8).

Clotting activities were determined using the pIMCU method.

TABLE 8 Enzymatic activities of camel chymosin variants 309-323. Numbersare given in % cleavage of non-glycosylated camel chymosin (CamUGly).Clotting Proteolytic variant mutations (C) (P) C/P CamUGly N100Q N291Q100 100 100 309 R242E Q280E N100Q N291Q 133 59 225 310 R242E N252D N100QN291Q 136 63 216 311 N252D Q280E N100Q N291Q 108 96 112 312 Y243E Q280EN100Q N291Q 112 71 158 313 Y243E N252D N100Q N291Q 91 77 117 314 R254EQ280E N100Q N291Q 106 84 127 315 S273D Q280E N100Q N291Q 77 51 150 316R242E G251D N100Q N291Q 107 72 148 317 R242E G251D Q280E N100Q N291Q 13884 164 318 R242E S273D Q280E N100Q N291Q 136 98 139 319 N252D S273DQ280E N100Q N291Q 115 67 171 320 G251D S273D Q280E N100Q N291Q 114 64176 321 R242E R254E Q280E N100Q N291Q 134 66 202 322 R242E R254E S273DQ280E N100Q N291Q 126 60 211 323 Y243E R254E S273D N100Q N291Q 103 71144

All variants shown in table 8 reveal increased C/P ratios compared tononglycosylated camel chymosin. Several of these variants (309, 310,321, 322, 323) had even higher C/P than the best variant with singlenegative charge mutation (286). It is concluded that the C/P-increasingeffect, caused by introducing negative charges into the P10-P4interacting region on the chymosin structure, can be further enhanced bycombinations of the respective mutations.

Structure-Based Variations in Bovine Chymosin

Variants of bovine chymosin (SEQ ID NO:1) were made with amino acidchanges in positions determined by protein structural analysis (Table9). Mutations N252Q and N291Q were introduced into both N-glycosylationsites of these variants and the reference bovine chymosin (BovUGly) toyield non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the pIMCU method.

TABLE 9 Enzymatic activities of bovine chymosin variants 325-346.Numbers are given in % cleavage of non-glycosylated bovine chymosin(BovUGly). Clotting Proteolytic variant mutations (C) (P) C/P BovUGlyN252Q N291Q 100 100 100 325 V223F N252Q N291Q 54 130 41 327 A117S N252QN291Q 75 76 96 329 Q242R N252Q N291Q 76 166 45 330 Q278K N252Q N291Q 94112 83 332 H292N N252Q N291Q 96 71 133 333 Q294E N252Q N291Q 99 79 123334 K295L N252Q N291Q 106 182 58 335 D249N N252Q N291Q 89 129 68 337G244D N252Q N291Q 100 106 93 338 Q56H N252Q N291Q 110 140 77 339 L32IN252Q N291Q 86 124 69 340 K71E N252Q N291Q 44 50 86 341 P72T N252Q N291Q103 172 59 342 Q83T N252Q N291Q 92 103 88 343 V113F N252Q N291Q 42 44 95344 E133S N252Q N291Q 93 199 46 345 Y134G N252Q N291Q 106 115 91 346K71A N252Q N291Q 79 131 60

The data in table 9 demonstrate that variations Q56H, Y134G, and K295Llead to increased specific clotting activity and variations H292N andQ294E result in enhanced C/P ratios. Both H292 and Q294 are located in aloop partially covering the substrate binding cleft (FIG. 3), whichexplains the observed impact of the respective mutations in thesepositions on casein substrate specificity (C/P). Notably, whilesubstitutions H292N increased C/P and D249N as well as K295L decreasedC/P of bovine chymosin, inverse effects on C/P were observed by therespective reverse mutations N292H, N249D, and L295K in camel chymosin(Tab. 7). This demonstrates that these amino acid changes exert similareffects on chymosin specificity across species.

Variations of the Camel Chymosin N-Terminus

Variants of camel chymosin (SEQ ID NO:2) were made with amino acidchanges in positions determined by protein structural analysis of themolecular interactions of the N-terminal sequence Y11-D13 within thesubstrate binding cleft (Tab. 10). Mutations N100Q and N291Q wereintroduced into both N-glycosylation sites of these variants and thereference camel chymosin (CamUGly) to yield non-glycosylated,homogeneous protein samples.

Clotting activities were determined using the pIMCU method.

TABLE 10 Enzymatic activities of camel chymosin variants 347-366.Numbers are given in % cleavage of non-glycosylated camel chymosin(CamUGly). Clot- Proteo- ting lytic variant mutations (C) (P) C/PCamUGly N100Q N291Q 100 100 100 347 Y11H N100Q N291Q 76 131 58 348 Y11KN100Q N291Q 63 82 76 349 Y11R N100Q N291Q 55 277 20 350 Y11H D290E N100QN291Q 74 105 71 351 Y11R D290E N100Q N291Q 62 101 62 352 Y11F N100QN291Q 91 146 62 353 Y11I N100Q N291Q 96 83 116 354 Y11L N100Q N291Q 79108 74 355 Y11V N100Q N291Q 101 64 157 356 L12F N100Q N291Q 96 147 66357 L12I N100Q N291Q 83 91 91 359 D13N N100Q N291Q 88 131 67 360 D13QN100Q N291Q 100 169 59 361 D13S N100Q N291Q 88 164 54 362 D13T N100QN291Q 62 89 70 363 D13F N100Q N291Q 73 155 48 364 D13L N100Q N291Q 82196 42 365 D13V N100Q N291Q 49 86 57 366 D13Y N100Q N291Q 74 99 75

Analysis of the camel chymosin structure guided variations in theN-terminal sequence Y11-D13 as well as in position D290, a potentialinteraction partner of Y11 (FIG. 4). Since casein substrates competewith the N-terminal chymosin sequence for binding within the bindingcleft, amino acid substitutions that change interactions between bindingcleft and the motif Y11-D13 are expected to impact enzymatic activitytoward various casein substrates and, thus, the C/P ratio. The resultsof the respective variants 347-366 show strong variation of bothspecific clotting activity and C/P. Notably, variants 353 and 355 revealincreased C/P ratios. It is therefore concluded that amino acidsubstitutions Y11I and Y11V result in increased C/P ratios. Since thechymosin binding cleft consists mainly of hydrophobic amino acids(reference 9), both mutations might enhance binding of the N-term in thebinding cleft by improved hydrophobic interactions and, thus, inhibitnon-specific binding and hydrolysis of caseins (P).

Multi-Substitution Library 3

Another set of camel chymosin variants, each having multiplesubstitutions compared to wild type, were generated and analyzed asdescribed above. All variants have an amino acid sequence identical tocamel chymosin (SEQ ID NO:2), except for the variations mentioned in thetable. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the pIMCU method.

TABLE 11 Enzymatic activities of camel chymosin variants 367-416.Numbers are given in % cleavage of wild type camel chymosin (CHY-MAX M).Clotting Proteolytic variant (C) (P) C/P CHY-MAX M mutations 100 100 100367 R67Q N100Q L130I M157L V248I N291Q 46 64 72 368 N100Q L130I S132AM157L K231N 87 104 83 369 R67Q I96L L130I M157L L222I M256L 49 56 88 370R67Q L130I S132A M157L R242E V248I 23 32 70 371 R67Q N100Q M157L R242EM256L 100 62 162 372 R67Q G70D M157L R242E V248I 89 32 276 373 V32L R67QM157L L222I R242E 64 63 102 374 Y11V R67Q M157L V248I M256L 71 45 158375 R67Q V136I M157L L222I V248I 88 20 449 376 L130I M157L V248I M256LN291Q 90 80 112 377 R67Q I96L L130I M157L K231N R242E 124 37 339 378V32L R67Q L130I M157L L222I K231N 52 103 51 379 L130I V136I M157L L222IN292H 55 47 118 380 R67Q G70D M157L L222I N291Q 117 34 339 381 V32L R67QL130I K231N N292H 58 66 87 382 Y11V R67Q N100Q L130I V136I M157L 60 55109 383 R67Q L130I L222I R242E M256L 78 27 290 384 R67Q M157L L222IV248I N292H 83 97 86 385 V32L R67Q M157L M256L N291Q 85 143 60 386 R67QL130I S132A M157L L222I N292H 78 133 58 387 R67Q N100Q L130I M157L K231NN291Q 59 70 84 388 R67Q L130I K231N V248I N291Q 91 87 105 389 Y11V R67QL130I M157L L222I K231N 63 47 134 390 I45V L130I M157L K231N R242E 10843 253 391 V32L R67Q V136I M157L N291Q 104 84 124 392 R67Q N100Q L130ID158S V248I 70 67 105 393 I45V R67Q L130I M157L L222I K231N 79 54 147394 V32L R67Q L130I S132A M157L V248I 74 130 57 395 Y11V R67Q L130IM157L N291Q N292H 74 83 90 396 R67Q N100Q L130I M157L L222I K231N 60 8174 397 I45V R67Q G70D L130I S132A 68 75 90 398 I45V R67Q L130I V248IN292H 53 81 66 399 Y11V R67Q L130I M157L L222I R242E 106 28 373 400 R67QN100Q D158S L130I M157L L222I 57 58 98 401 R67Q L130I V136I M157L K231NV248I 71 79 89 402 I45V R67Q L130I L222I N291Q 91 89 103 403 R67Q G70DL130I M157L K231N M256L 89 53 167 404 V32L R67Q L130I M157L D158S V248I58 82 71 405 R67Q L130I M157L D158S R242E N291Q 92 16 556 406 R67Q L130IM157L D158S K231N N292H 53 74 72 407 R67Q L130I V248I M256L N292H 58 10755 408 V32L R67Q I96L L130I M157L V248I 35 76 46 409 R67Q I96L N100QL130I M157L N292H 96 36 263 410 V32L R67Q G70D N100Q M157L 68 66 104 411V32L R67Q L130I M157L K231N M256L 102 18 574 412 R67Q I96L M157L L222IK231N 120 55 220 413 R67Q M157L L222I K231N V248I 124 46 268 414 R67QL130I M157L R242E M256L N292H 115 59 196 415 R67Q L222I K231N V248I 8267 123 416 R67Q S132A L222I K231N R242E V248I 129 42 306

In table 11 are shown camel chymosin variants with data on specificclotting activity (C), unspecific proteolytic activity (P) as well asthe C/P ratio. Out of 50 variants 6 reveal between 10% and 29% increasedspecific clotting activity compared to wild type camel chymosin (CHY-MAXM). While 23 variants have more than 10% increased C/P ratios, the bestone, 411, shows a ca. 6× improvement compared to wild type camelchymosin (CHY-MAX M).

Mutational Analysis of Multi-Substitution Library 3

A statistical analysis of the positional and mutational effects onclotting activity (C) and the C/P ratio was performed based on theproteolytic data of library 3. The most beneficial mutations forincreased clotting and C/P are shown in tables 12 and 13, respectively.

TABLE 12 Mutational contributions (mean) to increased clotting activityand standard deviations (sd) based on statistical analysis. mutationmean sd R242E 4.63E−01 4.21E−02 I96L 2.31E−01 4.82E−02 N291Q 1.67E−013.97E−02 K231N 1.34E−01 3.52E−02 M256L 1.28E−01 4.44E−02 S132A 1.04E−013.35E−02 M157L 7.99E−02 3.49E−02

Based on the results shown in table 12 it is concluded that mutationsI96L, S132A, M157L, K231N, R242E, M256L, and N291Q increase the specificclotting activity of chymosin. It can consequently be expected thatthese mutations enable a lower dosing of chymosin in cheesemanufacturing.

TABLE 13 Mutational contributions (mean) to increased C/P ratio andstandard deviations (sd) based on statistical analysis. mutation mean sdR242E 6.66E−01 4.23E−02 G70D 3.32E−01 5.72E−02 Y11V 2.06E−01 3.61E−02K231N 1.45E−01 2.92E−02 L222I 1.09E−01 3.71E−02 V136I 1.02E−01 4.53E−02I96L 9.84E−02 6.02E−02 N291Q 4.78E−02 4.20E−02

Based on the results shown in table 13 it is concluded that mutationsY11V, G70D, I96L, V1361, L222I, K231N, R242E, and N291Q increase the C/Pratio of chymosin. It can consequently be expected that these mutationsresult in increased yields during cheese manufacturing using therespective chymosin variants.

Multi-Substitution Library 4

Another set of camel chymosin variants, each having multiplesubstitutions compared to wild type, were generated and analyzed asdescribed above. All variants have an amino acid sequence identical tocamel chymosin (SEQ ID NO:2), except for the variations mentioned in thetable. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the REMCAT method.

TABLE 14 Enzymatic activities of camel chymosin variants 417-461.Numbers are given in % cleavage of wild type camel chymosin (CHY-MAX M).Clotting Proteolytic variant (C) (P) C/P CHY-MAX M mutations 100 100 100417 Y11V K19T D59N S164G L166V L222I R242E N249E G251D 132 20 651 418Y11V K19T D59N I96L S164G L166I L222I R242E N249E G251D 114 21 556 419Y11I K19T D59N I96L S164G L166V L222I R242E N249E G251D 108 20 554 420Y11I K19T D59N I96L S164G L166I L222I R242E G251D 98 11 898 421 Y11VK19T D59N I96L L166V L222V R242E N249E G251D L253I 132 84 156 422 Y11VK19T D59N I96L S164G L166V R242E 105 13 802 423 Y11V K19T D59N I96LS164G L222V R242E G251D 89 8 1131 424 Y11V K19T D59N I96L S164G L166IR242E N249E G251D L253I 93 8 1111 425 Y11V K19T D59N I96L S164G L166VL222V R242E N249E G251D 105 18 572 426 Y11V K19T D59N I96L S164G L166IL222V R242E N249E G251D L253I 93 18 512 427 Y11V K19T D59N L166V L222IR242E N249E G251D L253I 137 42 323 428 Y11V K19T D59N I96L S164G L166VL222I R242E N249E 120 15 803 429 Y11V K19T D59N S164G L166I L222I R242EG251D 107 17 630 430 Y11V K19T D59N I96L S164G R242E G251D 89 11 801 431Y11V D59N I96L S164G L166I L222V R242E G251D L253I 79 28 283 432 Y11VD59N I96L S164G L166I L222I R242E G251D 102 24 432 433 Y11I D59N I96LS164G L166V L222V R242E G251D L253I 97 25 392 434 Y11V K19T D59N I96LS164G L222I R242E N249E G251D 99 33 301 435 Y11V K19T D59N I96L S164GL166I L222V R242E G251D 88 17 514 436 Y11V K19T D59N I96L S164G L166VL222V R242E N249E L253I 95 10 949 437 Y11V K19T D59N I96L S164G L166IL222V R242E N249E G251D 114 22 520 438 Y11I K19T I96L S164G L166V R242EN249E G251D 93 7 1262 439 Y11V K19T D59N I96L S164G L166V L222V R242EG251D 108 26 423 440 Y11V K19T D59N I96L S164G L222V R242E N249E G251D105 9 1196 441 Y11I K19T L222V R242E N249E G251D 122 26 469 442 Y11VK19T I96L L222V R242E N249E G251D 105 21 503 443 Y11I K19T D59N I96LS164G L166V L222V R242E N249E G251D 105 18 595 444 Y11V K19T I96L S164GL166V L222V R242E N249E G251D 96 8 1242 445 Y11I K19T D59N I96L S164GL166I L222V R242E N249E G251D 82 12 707 446 Y11I I96L S164G L166V L222VR242E N249E G251D 95 16 579 447 Y11I K19T D59N I96L S164G L222V R242EN249E 90 11 790 448 Y11I K19T D59N I96L L222V R242E N249E G251D 153 40381 449 Y11I K19T D59N I96L S164G L222I R242E 89 16 564 450 Y11I K19TD59N I96L S164G L166V R242E G251D 88 5 1686 451 Y11I K19T D59N S164GL166I L222V R242E G251D 93 21 440 452 Y11I I96L L222V R242E N249E G251D122 22 566 453 Y11I I96L S164G L222I R242E 74 5 1375 454 Y11V K19T I96LL166V L222V R242E G251D 119 52 228 455 Y11I D59N I96L S164G L222I R242EG251D 105 9 1139 456 Y11I D59N I96L S164G L222V R242E N249E G251D 95 15615 457 Y11I K19T D59N I96L S164G L222I R242E N249E G251D 101 7 1419 458Y11I D59N I96L S164G L166V L222V R242E G251D 89 16 572 459 Y11V K19TD59N I96L L222V R242E G251D 143 62 230 460 Y11I K19T S164G L166I L222VR242E N249E G251D 80 13 625 461 Y11I D59N I96L S164G L166V L222V R242EN249E G251D 96 35 273

In table 14 are shown camel chymosin variants with data on specificclotting activity (C), unspecific proteolytic activity (P) as well asthe C/P ratio. Out of 45 variants 11 reveal between 14% and 53%increased specific clotting activity compared to wild type camelchymosin (CHY-MAX M). While all 45 variants have more than 10% increasedC/P ratios, the best one, 450, shows a ca. 17× improvement compared towild type camel chymosin (CHY-MAX M).

Mutational Analysis of Multi-Substitution Library 4

A statistical analysis of the positional and mutational effects onclotting activity (C) and the C/P ratio was performed based on theproteolytic data of library 4. The most beneficial mutations forincreased clotting and C/P are shown in tables 15 and 16, respectively.

TABLE 15 Mutational contributions (mean) to increased clotting activityand standard deviations (sd) based on statistical analysis. mutationmean sd D59N 3.99E−01 3.48E−02 L222I 2.05E−01 2.64E−02 L166V 1.92E−012.39E−02 N249E 1.45E−01 1.88E−02 G251D 9.79E−02 2.29E−02 Y11V 8.54E−021.56E−02 R242E 5.14E−02 2.06E−02

Based on the results shown in table 15 it is concluded that mutationsY11V, D59N, L166V, L222I, R242E, N249E, and G251D increase the specificclotting activity of chymosin. It can consequently be expected thatthese mutations enable a lower dosing of chymosin in cheesemanufacturing.

TABLE 16 Mutational contributions (mean) to increased C/P ratio andstandard deviations (sd) based on statistical analysis. mutation mean sdS164G 7.51E−01 4.50E−02 K19T 2.85E−01 4.93E−02 I96L 2.43E−01 4.16E−02R242E 2.25E−01 7.12E−02 L253I 2.22E−01 4.61E−02 Y11I 1.30E−01 4.93E−02N249E 9.52E−02 3.86E−02 Y11V 9.49E−02 3.55E−02

Based on the results shown in table 16 it is concluded that mutationsY11I, Y11V, K19T, I96L, S164G, R242E, N249E, and L253I increase the C/Pratio of chymosin. It can consequently be expected that these mutationsresult in increased yields during cheese manufacturing using therespective chymosin variants.

Selected variants from multi-substitution library 4 were fermented againin 70L followed by purification and characterization regarding theirproteolytic profile (table 17).

TABLE 17 Enzymatic activities of selected camel chymosin variants from70 L fermentation. Numbers are given in % cleavage of wild type camelchymosin (CHY-MAX M). Clotting Proteolytic variant (C) (P) C/P CHY-MAX Mmutations 100 100 100 433 Y11I D59N I96L S164G L166V L222V R242E G251DL253I 151 11 1356 436 Y11V K19T D59N I96L S164G L166V L222V R242E N249EL253I 188 9 2007 453 Y11I I96L S164G L222I R242E 153 8 1893 457 Y11IK19T D59N I96L S164G L222I R242E N249E G251D 217 7 3002

In table 17 are shown camel chymosin variants from 70L fermentation withdata on specific clotting activity (C), unspecific proteolytic activity(P) as well as the C/P ratio. All 4 variants reveal between 51% and 117%increased specific clotting activity compared to wild type camelchymosin (CHY-MAX M). While all 4 variants have more than 13-foldincreased C/P ratios, the best one, 457, shows a ca. 30× improvementcompared to wild type camel chymosin (CHY-MAX M).

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1-15 (canceled)
 16. An isolated chymosin polypeptide variant comprisingan alteration in one or more amino acid positions relative to a parentpolypeptide having chymosin activity, wherein the alteration comprise asubstitution, a deletion, or an insertion at amino acid position M157 ofthe parent polypeptide, and a further alteration selected from asubstitution, deletion, or insertion at one or more amino acid positionsof the parent polypeptide selected from G70, 5132, V136, L222, K231,N291, wherein (i) the amino acid position of the parent polypeptide isdetermined by alignment of the parent polypeptide with the polypeptideof SEQ ID NO:2 (camel chymosin), (ii) the parent polypeptide has atleast 80% amino acid sequence identity with SEQ ID NO: 2, (iii) thevariant has fewer than 30 amino acid alterations as compared to SEQ IDNO: 2, as determined by an alignment of the amino acid sequence of thevariant with the amino acid sequence of SEQ ID NO: 2, and (iv) thevariant has one or both of (i) a higher ratio of specific clottingactivity to proteolytic activity (“C/P ratio”) and (ii) at least 85% ofthe specific clotting activity of its parent polypeptide.
 17. Theisolated chymosin polypeptide variant of claim 16, having an additionalalteration in its amino acid sequence relative to the parent polypeptideselected from a substitution, deletion, or insertion at one or more ofamino acid positions Y11, K19, Y21, Q56, D59, S74, H76, I96, S164, L166,8242, Y243, N249, G251, N252, 8254, M256, 5273, Q280, F282, L295, andV309, of the parent polypeptide, wherein the amino acid position of theparent polypeptide is determined by alignment of the parent polypeptidewith the polypeptide of SEQ ID NO:2.
 18. The isolated chymosinpolypeptide variant of claim 16, having an additional alteration in itsamino acid sequence relative to the parent polypeptide selected from asubstitution, deletion, or insertion at one or more of amino acidpositions V32, R67, N100, and L130 of the parent polypeptide, whereinthe amino acid position of the parent polypeptide is determined byalignment of the parent polypeptide with the polypeptide of SEQ ID NO:2.19. The isolated chymosin polypeptide variant of claim 16, wherein thealteration at position M157 is the substitution M157L.
 20. The isolatedchymosin polypeptide variant according to claim 16, wherein the furtheralteration is selected from one or more of S132A, G70D, V136I, L222I,K231N, and N291Q.
 21. The isolated chymosin polypeptide variantaccording to claim 17, wherein the additional alteration is selectedfrom one or more of Y11I, Y11V, K19T, Y21S, Q56H, D59N, S74D, H76Q,I96L, S164G, L166V, R242E, R242D, Y243E, N249E, N249D, G251D, N252D,R254E, M256L, S273D, S273Y, Q280E, F282E, L295K, and V309I.
 22. Theisolated chymosin polypeptide variant according to claim 18, wherein theadditional alteration is selected from one or more of V32L, R67Q, N100Q,and L130I.
 23. An isolated chymosin polypeptide variant according toclaim 16, wherein the alteration comprises substitutions selected from:N100Q, L130I, S132A, M157L, and K231N; R67Q, G70D, M157L, L222I, andN291Q; and V32L, R67Q, V1361, M157L, and N291Q.
 24. An isolated chymosinpolypeptide variant according to claim 16, wherein the alterationcomprises substitutions selected from: R67Q, I96L, L130I, M157L, K231N,and R242E; R67Q, M157L, L222I, K231N, and V248I; R67Q, I96L, M157L,L222I, and K231N; R67Q, N100Q, M157L, R242E, M256L; R67Q, G70D, M157L,R242E, and V248I; V32L, R67Q, M157L, L222I, and R242E; Y11V, R67Q,M157L, V248I, and M256L; R67Q, V136I, M157L, L222I, and V248I; L130I,M157L, V248I, M256L, and N291Q; L130I, V136I, M157L, L222I, and N292H;Y11V, R67Q, N100Q, L130I, V136I, and M157L; V32L, R67Q, M157L, M256L,and N291Q; Y11V, R67Q, L130I, M157L, L222I, and K231N; I45V, L130I,M157L, K231N, and R242E; I45V, R67Q, L130I, M157L, L222I, and K231N;Y11V, R67Q, L130I, M157L, L222I, and R242E; R67Q, G70D, L130I, M157L,K231N, and M256L; R67Q, L130I, M157L, D158S, R242E, and N291Q; R67Q,I96L, N100Q, L130I, M157L, and N292H; V32L, R67Q, G70D, N100Q, andM157L; V32L, R67Q, L130I, M157L, K231N, and M256L; and R67Q, L130I,M157L, R242E, M256L, and N292H.
 25. A method for making an isolatedchymosin polypeptide variant according to claim 16, comprising: makingan alteration at one or more positions in a parent polypeptide havingchymosin activity, wherein the alteration comprise a substitution, adeletion, or an insertion at amino acid position M157 of the parentpolypeptide, and a further alteration selected from a substitution,deletion, or insertion at one or more amino acid positions of the parentpolypeptide selected from G70, S132, V136, L222, K231, N291, to obtainthe variant; wherein: (i) the amino acid position of the parentpolypeptide is determined by alignment of the parent polypeptide withSEQ ID NO:2 (camel chymosin), (ii) the parent polypeptide has at least80% amino acid sequence identity with SEQ ID NO: 2, and (iii) thevariant has fewer than 30 amino acid alterations as compared to SEQ IDNO: 2, as determined by an alignment of the amino acid sequence of thevariant with the amino acid sequence of SEQ ID NO:
 2. 26. The methodaccording to claim 25, further comprising making an additionalalteration in the amino acid sequence of the variant relative to theparent polypeptide selected from a substitution, deletion, or insertionat one or more of amino acid positions Y11, K19, Y21, Q56, D59, S74,H76, 196, S164, L166, R242, Y243, N249, G251, N252, R254, M256, 5273,Q280, F282, L295, and V309, of the parent polypeptide, wherein the aminoacid position of the parent polypeptide is determined by alignment ofthe parent polypeptide with the polypeptide of SEQ ID NO:2.
 27. Themethod according to claim 25, further comprising making an additionalalteration in the amino acid sequence of the variant relative to theparent polypeptide selected from a substitution, deletion, or insertionat one or more of amino acid positions V32, R67, N100, and L130 of theparent polypeptide, wherein the amino acid position of the parentpolypeptide is determined by alignment of the parent polypeptide withthe polypeptide of SEQ ID NO:2.
 28. The method according to claim 25,wherein the alteration at position M157 is the substitution M157L. 29.The method according to claim 25, wherein the further alteration isselected from one or more of S132A, G70D, V136I , L222I, K231N, andN291Q.
 30. The method according to claim 26, wherein the additionalalteration is selected from one or more of Y11I, Y11V, K19T, Y21S, Q56H,D59N, S74D, H76Q, I96L, S164G, L166V, R242E, R242D, Y243E, N249E, N249D,G251D, N252D, R254E, M256L, S273D, S273Y, Q280E, F282E, L295K, andV309I.
 31. The method according to claim 17, wherein the additionalalteration is selected from one or more of V32L, R67Q, N100Q, and L130I.32. The method according to claim 25, wherein the alteration comprisessubstitutions selected from: N100Q, L130I, S132A, M157L, and K231N;R67Q, G70D, M157L, L222I, and N291Q; and V32L, R67Q, V136I, M157L, andN291Q.
 33. The method according to claim 25, wherein the alterationcomprises substitutions selected from: R67Q, I96L, L130I, M157L, K231N,and R242E; R67Q, M157L, L222I, K231N, and V248I; R67Q, I96L, M157L,L222I, and K231N; R67Q, N100Q, M157L, R242E, M256L; R67Q, G70D, M157L,R242E, and V248I; V32L, R67Q, M157L, L222I, and R242E; Y11V, R67Q,M157L, V248I, and M256L; R67Q, V136I, M157L, L222I, and V248I; L130I,M157L, V248I, M256L, and N291Q; L130I, V1361, M157L, L222I, and N292H;Y11V, R67Q, N100Q, L130I, V136I, and M157L; V32L, R67Q, M157L, M256L,and N291Q; Y11V, R67Q, L130I, M157L, L222I, and K231N; I45V, L130I,M157L, K231N, and R242E; I45V, R67Q, L130I, M157L, L222I, and K231N;Y11V, R67Q, L130I, M157L, L222I, and R242E; R67Q, G70D, L130I, M157L,K231N, and M256L; R67Q, L130I, M157L, D158S, R242E, and N291Q; R67Q,I96L, N100Q, L130I, M157L, and N292H; V32L, R67Q, G70D, N100Q, andM157L; V32L, R67Q, L130I, M157L, K231N, and M256L; and R67Q, L130I,M157L, R242E, M256L, and N292H.
 34. A method for making a food or feedproduct, comprising adding an effective amount of the isolated chymosinpolypeptide variant according to claim 16 to the food or feedingredient(s).
 35. A method according to claim 34, wherein the food orfeed product is a milk-based product.
 36. A method according to claim34, wherein the food or feed product is a cheese.
 37. A method accordingto claim 36, wherein the cheese is selected from Pasta filata, Cheddar,and Continental-type cheeses.
 38. A method according to claim 36,wherein the cheese is Soft Cheese or White Brine Cheese.
 39. A food orfeed product comprising a chymosin polypeptide variant according toclaim 16.